Alzheimer's disease (AD), Parkinson's disease (PD) and Lewy Body disease (LBD) are the most commonly found neurodegenerative disorders in the elderly. Although their incidence continues to increase, creating a serious public health problem, to date these disorders are neither curable nor preventable. There is a genetic component to these diseases, however most cases arise spontaneously in the population in the absence of mutations. Nerve damage results from the development of protein aggregates composed of proteins normally expressed in the brain. It is not known what causes the conversion of normally non-toxic proteins to their toxic state.
Recent epidemiological studies have demonstrated a close clinical relationship between AD and PD, as about 30% of Alzheimer's patients also have PD. Compared to the rest of the aging population, patients with AD are more likely to develop concomitant PD. Furthermore, PD patients that become demented usually have developed classical AD.
Although each neurodegenerative disease appears to have a predilection for specific brain regions and cell populations, resulting in distinct pathological pictures, PD, AD and LBD also share common pathological hallmarks. Patients with familial AD, Down syndrome and sporadic AD develop Lewy bodies on the amygdala which are the classical neuropathological hallmarks of PD. Additionally, each disease is associated with the degeneration of neurons, interneuronal synaptic connections and eventually cell death, the depletion of neurotransmitters, and abnormal accumulation of misfolded proteins, the precursors of which participate in normal central nervous system function.
Biochemical studies have confirmed the link between AD, PD and LBD. The central component of Lewy bodies is α-synuclein, also known as NACP (Non-amyloid component precursor). A proteolytic fragment of NACP, known as NAC, is a key component of amyloid plaques in AD. This suggests that α-synuclein plays a role in the pathogenesis of both AD and PD.
These findings have lead to the development of a new classification of neurodegenerative disorders denominated Lewy body disease (LBD). Patients with LBD are characterized by dementia, parkinsonism, psychiatric alterations, deposition of amyloid, and formation of Lewy bodies (LBs) with filamentous characteristics. LBs are the pathogenic hallmark of both PD and LBD. Although there are several animal models that mimic some aspects of AD and others that mimic some aspects of PD, there are no models that combine the characteristics of AD and PD, as seen in LBD.
The neuritic plaques that are the classic pathological hallmark of AD are composed essentially of Aβ, a 39-43 amino acid (aa) proteolytic product of the Alzheimer's amyloid precursor protein (APP), and NAC, a 35 aa proteolytic fragment of the NACP protein. Both Aβ and NAC were first identified in amyloid plaques as proteolytic fragments of their full length proteins, for which the full length cDNAs were identified and cloned. The cloning of APP (Kang et al., 1987) lead to a burst of research in which a number of mutations in AD were found that were associated with familial forms of AD including mutations at K670N/M671L (Swedish mutation), V717I (London mutation), V717F (Indiana mutation), presenilin 1 and presinilin 2. (Note, mutations are denoted by the wild type amino acid, the number of the amino acid at which the mutation is found and the amino acid that is found in the mutant form of the protein.) These mutations seem to alter the processing of APP preferentially to the Aβ1-42 proteolytic fragment, which has a propensity to form aggregates that are pathogenic. However, such mutations cannot account for the majority of Alzheimer's patients in whom the disease arises spontaneously.
APP is expressed abundantly in synapses under normal conditions, is well conserved across species, and has been implicated in neural plasticity, learning, and memory. APP has three alternative splice variants in which exons 7 and 8 (hAPP695), exon 8 (hAPP751) or no exons (hAPP770) are spliced out of the full length transcript. The ratio of the three forms varies between regions of the brain and in normal vs. disease states; however, no definitive pattern of normal vs. abnormal has been determined (Rockenstein et al., 1995). It is not known what regulates the differential splicing of the transcript, but evidence suggests that splicing within neurons can be regulated by factors that influence neuronal differentiation and activity. Additionally, APP is processed into proteolytic fragments Aβ39-43 under normal conditions. Disease results from an imbalance in the production of the fragments, biasing the overproduction of the proteolytic fragments, especially those that can initiate the formation of aggregates (i.e. Aβ1-42).
α-Synuclein is part of a large family of proteins including β- and γ-synuclein and synoretin. As with APP, α-synuclein is expressed in the normal state in synapses and is believed to play a role in neural plasticity, learning and memory. Mutations in human (h)α-synuclein that enhance the aggregation of α-synuclein have been identified (A30P and A53T) and are associated with rare forms of autosomal dominant forms of PD. The mechanism by which these mutations increase the propensity of α-synuclein to aggregate are unknown.
Despite the fact that a number of mutations can be found in APP and α-synuclein in the population, most cases of AD and PD arise spontaneously. The most frequent sporadic forms of these diseases are associated with an abnormal accumulation of Aβ and α-synuclein, respectively. However, the reasons for overaccumulation of these proteins is unknown. Aβ is secreted from neurons and accumulates in extracellular amyloid plaques. Additionally Aβ can be detected inside neurons. α-Synuclein accumulates in intraneuronal inclusions called Lewy bodies. Although the two proteins are typically found together in extracellular neuritic AD plaques, they are also occasionally found together in intracellular inclusions.
Studies have been performed to analyze the processing of the non-toxic precursor proteins to their toxic proteolytic products, both in tissue culture systems and using purified proteins. Aggregates may be formed in nerve cell cultures by overexpression of α-synuclein. Overexpression of wild-type APP does not cause formation of extracellular protein aggregates in culture; however, overexpression of the Swedish APP mutation, the most pathogenic of the APP mutations, does. Aggregates may also be formed in response to oxidative stress in cell culture in cells overexpressing α-synuclein, suggesting that it may be important in the development of disease. Additionally, purified Aβ and NAC peptides are able to form aggregates in vitro, either alone or mixed, under identical conditions of temperature and pH.
Attempts to establish animal models of AD, PD and LBD have been disappointing. Initially, animals expressing transgenic APP failed to show extensive AD-type neuropathology (Kammesheidt et al., 1992; Lamb et al., 1993; Mucke et al., 1994; Higgins et al., 1995; Andrää et al., 1996). This was likely due to low levels of protein expression. Games et al. (1995) were able to generate a transgenic mouse that showed some AD-type pathology due to the high level of expression of a mutant APP (V717F) driven by a PDGF-β promoter. The transgenic animals did exhibit deposits of human Aβ in the hippocampus, corpus callosum and the cerebral cortex, but in no other regions of the brain. Plaques were observed, but there were no neurofibrilary tangles. The author stated that such results were expected as neurofibrilary tangles did not exist in rodents.
Transgenic mice expressing the Swedish double mutation (670/671) or the Swedish mutation in conjunction with the London mutation (V717I) under control of the Thy1 expression cassette were also generated (Sturchler-Pierrat et al., 1997). The age of development of plaques varied from 6 months to more than two years in each of the mice strains, and seemed to correlate well with expression levels of the proteins. Lower expression levels were required to induce pathological changes associated with disease in the animals carrying both the Swedish and Indiana mutations. However, neither animal was a full and accurate representation of the disease. Plaque formation accompanied by neuritic changes, dystrophic cholinergic fibers and inflammation were observed in the hippocampus and neocortex, but not in other brain regions. In a separate study using transgenic mice expressing the Swedish mutant form of APP under the PrP-promotor (Hsiao et al., 1996), mice were found to have normal learning and memory at 3 months, but showed impairment by 9 or 10 months. Plaques were observed in the cortical and limbic areas in these transgenic mice and a 14-fold increase in Aβ1-42/43 was observed.
As Aβ1-42 was known to be one of the strongly causative factors in AD, Mucke et al. (2000) created a transgenic mouse that expressed only the toxic fragment of APP, Aβ1-42. Expression of the Aβ1-42 fragment was toxic to the cells, but no plaques were formed, suggesting a plaque independent role for Aβ1-42 in the progression of AD.
Additionally, transgenic animals expressing APP were found to display a variety of neurological problems including learning deficits, disturbed behavior and seizures. Again, the severity of neurological dysfunction seemed to be tied to the level of expression of the APP protein. Differences could also be attributed to the regions of the brain in which APP was expressed due to differences in promoters, integration into the genome, etc. As with the physiological symptoms though, neurological dysfunction in these animals resembled some aspects, but not all, of those seen in AD, PD and LBD. The lack of an animal model for the diseases was clearly not due to a lack of effort, but instead due to an inability to induce all of the changes that occur in the disease state.
The effect of the expression of the transgenic sequence was also largely dependent on the strain of mice in which the mutant gene was expressed. For example, concentrations of APP that produce plaques in outbred transgenic lines were found to be lethal in inbred FVB/N or C57BL/6J mice (Carlson, et al., 1997). Lines of FVB/N mice expressing low enough levels of APP695 to be viable were found to develop a CNS disorder that included neophobia, impaired spatial alternation, with diminished glucose utilization and astrogliosis mainly in the cerebrum (Hsiao et al., 1995). A similar syndrome is known to occur in about 20% of non-transgenic mice of the same strain, suggesting that the APP promotes a tendency already present in the strain.
Attempts were made to generate more accurate models of AD by crossing strains of transgenic mice to alter the pathology of the disease. Overexpression of the fibroblast growth factor 2 gene (FGF2) in transgenic mice overexpressing APP made APP more lethal, but did not alter the symptoms seen. Holtzman et al (2000) generated a number of bigenic mice by crossing transgenic mice expressing the V717F mutant of APP with mice expressing various forms of apolipoprotein E (apoE). The ε4 allele of apoE was the first genetic risk factor identified for sporadic and late-onset familial AD (FAD) (Strittmafter et al., 1993). Later the ε3 allele of apoE was found to be a weaker risk factor for the disease. Expression of apoE mutants, especially the ε4 allele, together with the V717F mutant version of APP in mice increased the development of fibrillar deposits, neuritic plaques and neuritic degradation. Similar pathological hallmarks of AD were observed in mice expressing only the V717F mutant of APP, simply at an older age. The combination of the expression of the transgenes did not change the observed pathology of the disease, simply the time frame in which the pathology was observed. Expression of the V717F mutant version of APP in an apoE−/− knockout mouse resulted in an absence of neuritic degradation. This study demonstrated a clear role for ApoE in the pathology of AD, but did not provide a more accurate model of the disease than the singly transgenic mouse.
Transgenic mice expressing α-synuclein under control of the PDGFβ promotor have also been generated for use as a model of PD and LBD (Masliah et al., 2000). Neuronal expression of α-synuclein resulted in the progressive accumulation of α-synuclein and the development of inclusions in neurons in the neocortex, hippocampus and substantial nigra. Additionally electron-dense intranuclear deposits and cytoplasmic inclusions were observed. The mice demonstrated motor impairments which were associated with loss of dopaminergic terminals in the basal ganglia. However, no amyloid plaques, fibrillary tangles or cell death were observed.
A number of α-synuclein transgenic mice have also been generated. However, despite the use of different promoters (e.g. PDGFβ, Thy-1) as well as the use of both wild type and mutant forms (A30P and A53T) of α-synuclein, none of the mice provide a completely accurate model of PD. As with the various APP transgenic mice, differences were seen in the amount and location of protein expressed, depending on the promoter used and the site of insertion of the transgene into the genome. For example, when expression from the hα-synuclein transgene was driven by the PDGF-β promotor, the expression pattern of hα-synuclein most closely resembled that seen in normal brain or in diffuse LBD. Expression of hα-synuclein using a Thy-1 promoter resulted in higher expression of the transgene in a pattern more closely resembling the pattern of expression seen in PD. Higher expression resulted in more severe pathology. Mice expressing mutant versions of hα-synuclein developed hallmarks of disease earlier than those expressing the wild type version of the protein. Additionally, neurological dysfunction was dependent on the regions of the brain in which the protein was expressed.
Disease states are often the result of multiple changes in the organism, rather than a change in the expression level of a single protein or a single mutation within a gene. Although each of the transgenic animals described above had some hallmarks of AD, PD and/or LBD, none of them could serve as a complete model of any of the diseases. Not a single animal was found to have neurofibrillary tangles, a classic indicator of AD. The realization of the overlapping pathologies and occurrence of AD, PD and LBD emphasizes the need for an animal model that displays a more complete set of the pathologies and clinical manifestations of the diseases.